1. Field of the Invention
The present invention relates to the fields of oncology, genetics and molecular biology. More particular the invention relates to the identification, on human chromosome 11, of a tumor suppressor gene. Defects in this gene are associated with the development of cancers, such as lung carcinoma.
2. Description of Related Art
Oncogenesis was described by Foulds (1958) as a multistep biological process, which is presently known to occur by the accumulation of genetic damage. On a molecular level, the multistep process of tumorigenesis involves the disruption of both positive and negative regulatory effectors (Weinberg, 1989). The molecular basis for human colon carcinomas has been postulated, by Vogelstein and coworkers (1990), to involve a number of oncogenes, tumor suppressor genes and repair genes. Similarly, defects leading to the development of retinoblastoma have been linked to another tumor suppressor gene (Lee et al., 1987). Still other oncogenes and tumor suppressors have been identified in a variety of other malignancies. Unfortunately, there remains an inadequate number of treatable cancers, and the effects of cancer are catastrophicxe2x80x94over half a million deaths per year in the United States alone.
Lung cancer is one of the most fatal and frequent human cancers in the United States (Parker et al., 1997). Identification of the multiple tumor-suppressor genes involved in pathogenesis of lung cancer is a critical step in the development of new diagnostic methods and tumor-specific treatments. A large body of evidence suggests that chromosome 11 may harbor at least one tumor-suppressor gene(s) involved in lung cancer (Rasio et al., 1995) and a variety of other cancers including colon (Gustafson et al., 1994), breast (Carter et al., 1994), cervical (Hampton et al., 1994), head and neck (El-Naggar et al., 1996), ovarian cancers (Davis et al., 1996) and melanoma (Tomlinson et al., 1996). That this region of the chromosome has suppressor oncogene activity has been shown by the introduction of a normal chromosome 11, or a derivative t(X,11) chromosome containing 11pter-q23, into tumorigenic cells lines reversing the tumorigenic potential. Tumor suppressing activity has been demonstrated on chromosome 11q23 using lung (Satoh et al., 1993), Wilms tumor (Weissman et al., 1987), breast (Negrini et al., 1994) and cervical carcinoma cell lines (Saxon et al., 1986).
Since allelic loss of chromosome 11q22-24 has been implicated in a variety of cancers, significant effort has been applied to detailed mapping of this region of the genome using polymorphic markers. Using this approach, it has been possible to define a common and restricted region of loss of heterozygosity (LOH) region between markers STMY1 at 11q22 and ApoC3 at 11q23 in these neoplasms (Arai et al., 1996). These studies suggest the presence of functional tumor-suppressor gene(s) on chromosome 11q22-q24 localized centromeric to the t(X;11) translocation breakpoint at 11q23.
Despite all of this information, the identity of the gene (or genes) involved with the 11q23-related tumor suppression remains elusive. Without identification of a specific gene and deduction of the protein for which it codes, it is impossible to begin developing an effective therapy targeting this product. Thus, it is an important goal to isolate the tumor suppressor(s) located in this region and determine its structure and function.
In specific embodiments, the present invention provides an isolated polynucleotide comprising a region, or the complement thereof, encoding a tumor suppressor designated PPP2R1B or an allelic variant or mutant thereof. More particularly, the tumor suppressor coding region is selected from the group consisting of Xenopus, porcine and human. In yet more preferred embodiments, the tumor suppressor coding region is human. In those embodiments in which the polynucleotide is a mutant tumor suppressor, the mutant may comprise a deletion mutation, an insertion mutation, a frameshift mutation, a nonsense mutation, a missense mutation or splice mutation. In certain embodiments, the mutant is a splice mutant. More specifically, the splice mutation is in intron 8. In more defined embodiments, the splice mutation results from a mutation in intron 8.
Yet another preferred embodiments contemplated is one in which the mutation is a change from A1540 to G1540 as compared to the wild-type tumor suppressor. More particularly, the mutation results in a change from ASP to GLY at position 504 of the tumor suppressor. In specific embodiments, the mutation results in a change from a loss of heterozygosity. More specific embodiments contemplate a mutation that results in a change from G51 to C51 in the tumor suppressor. In yet another embodiment, the mutation results in a change from GLY to ARG at amino acid residue 8 of the tumor suppressor. In certain other embodiments, the mutation is a germline mutation. In yet another embodiments, the mutation is a change from G298 to A298 in the tumor suppressor. More particularly, the mutation results in a change from GLY to ASP at amino acid 90 of the tumor suppressor. In another contemplated embodiment, the mutation is a change from A1056 to G1056 in the tumor suppressor. Also contemplated is a mutation that results in a change from LYS to GLU at amino acid 343 of the tumor suppressor. In another example, the mutation is a change from C222 to T222 in the tumor suppressor. Yet a further mutation results in a change from PRO to SER at amino acid 65 of the tumor suppressor. In still another embodiments, the mutation is a change from T1663 to C1663 in the tumor suppressor. An additional mutation results in a change from VAL to ALA at amino acid 545 of the tumor suppressor. Another mutation is one in which there is a change from T331 to C331 in the tumor suppressor. Another specific embodiments is one in which the mutation results in a change from LEU to PRO at amino acid 101 of the tumor suppressor. Yet another mutation is a change from T1372 to C1372 in the tumor suppressor. More particularly, the mutation results in a change from VAL to ALA at amino acid 448 of the tumor suppressor. In specific embodiments, the mutation is an in-frame deletion of bases 717 to 1583. More particularly this mutation results in a truncated tumor suppressor expression. Yet more specifically, the truncated tumor suppressor lacks amino acids 230 to 518 of the wild-type tumor suppressor. In specific embodiments, the mutation is a deletion of nucleotides 1584 through to 1726. In other embodiments, the mutation results in a frameshift in tumor suppressor expression. In still another embodiments, the tumor suppressor has a frameshift between amino acids 519 and 601. In certain embodiments, the mutation is a deletion of nucleotides 1057 to 1191. More particularly, the mutation results in a truncated tumor suppressor expression. Specifically, the truncated tumor suppressor may lack amino acids 344 to 388 of the wild-type tumor suppressor. In other particularly preferred embodiments, the mutation is a deletion of nucleotides 1315 through to 1505. More particularly, the mutation results in a frameshift in tumor suppressor expression. Yet more particularly, the tumor suppressor has a frameshift between amino acids 422 and 601.
In specific embodiments, the tumor suppressor has the amino acid sequence of SEQ ID NO:1. In other embodiments, the tumor suppressor has the amino acid sequence of SEQ ID NO:2. In yet a further embodiments, the polynucleotide sequence comprises the coding sequence of SEQ ID NO:4 or the complement thereof. In certain specific embodiments, the polynucleotide sequence comprises a porcine PPP2R1B or the complement thereof. In other exemplary embodiments, the polynucleotide sequence comprises a Xenopus PPP2R1B or the complement thereof. In particularly defined embodiments, the polynucleotide may be selected from the group consisting of genomic DNA, complementary DNA and RNA. More particularly, the polynucleotide may be a complementary DNA and further comprise a promoter operably linked to the region, or the complement thereof, encoding the tumor suppressor. In still further embodiments, the polynucleotide further may comprise a polyadenylation signal operably linked to the region encoding the tumor suppressor. In additional embodiments the polynucleotide also may comprise an origin of replication. In specific embodiments, the polynucleotide is a viral vector selected from the group consisting of retrovirus, adenovirus, herpesvirus, vaccinia virus and adeno-associated virus. In further embodiments, the polynucleotide is packaged in a virus particle. In additional embodiments, the polynucleotide is packaged in a liposome.
In certain specific embodiments the polynucleotide is of a size selected from the group consisting of about 100 bases, about 200 bases, about 300 bases, about 400 bases about 500 bases, about 600 bases about 700 bases, about 800 bases, about 900 bases, about 1000 bases, about 1200 bases, about 1500 bases, about 1800 bases, about 1922 bases and about 2000 bases.
Also provided herein is an isolated polypeptide encoding a tumor suppressor designated as PPP2R1B. In specific embodiments the tumor suppressor has the amino acid sequence as set forth in SEQ ID NO:1. In other particular embodiments, the tumor suppressor has an amino acid sequence as set forth in SEQ ID NO:2. In still further embodiments, the tumor suppressor has an amino acid sequence as set forth in SEQ ID NO:3.
Also provided herein is an isolated peptide having between about 10 and about 50 consecutive residues of a tumor suppressor designated as PPP2R1B. It is contemplated that the peptide may be conjugated to a carrier molecule. More particularly, the carrier molecule is selected from the group consisting of KLH and BSA. In specific embodiments, the tumor suppressor may have the amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3 or any sequence variant thereof.
Also contemplated herein is a monoclonal antibody that binds immunologically to a tumor suppressor designated as PPP2R1B. In particularly embodiments the antibody does not bind immunologically to other human polypeptides. In other embodiments, the antibody binds to a non-human PPP2R1B and does not bind to human PPP2R1B. In some embodiments, the antibody further comprises a detectable label. More particularly, the label may be selected from the group consisting of a fluorescent label, a chemiluminescent label, a radiolabel and an enzyme.
Also contemplated is a hybridoma cell that produces a monoclonal antibody that binds immunologically to a tumor suppressor designated as PPP2R1B. Another embodiment contemplates a polyclonal antisera, antibodies of which bind immunologically to a tumor suppressor designated as PPP2R1B. In specific embodiments the antisera is derived from an animal other than human, pig or Xenopus.
The present invention further provides a method of diagnosing a cancer comprising the steps of obtaining a sample from a subject; and determining the expression a functional PPP2R1B tumor suppressor in cells of the sample. In specific embodiments, the cancer may be selected from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, pancreas, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood cancer. In more specific embodiments, the cancer is a lung cancer, colon cancer, breast cancer or cervical cancer. In preferred embodiments, the sample is a tissue or fluid sample. In certain embodiments, the determining comprises assaying for a nucleic acid from the sample. More particularly, the method further comprises subjecting the sample to conditions suitable to amplify the nucleic acid. Alternative embodiments contemplate that the determining comprises contacting the sample with an antibody that binds immunologically to a PPP2R1B. More specifically, the method further comprises subjecting proteins of the sample to ELISA. In other embodiments, the method further comprises the step of comparing the expression of PPP2R1B with the expression of PPP2R1B in non-cancer samples. In specific embodiments, the comparison involves evaluating the level of PPP2R1B expression. In other embodiments, the comparison involves evaluating the structure of the PPP2R1B gene, protein or transcript. More particularly, the evaluating is an assay selected from the group consisting of sequencing, wild-type oligonucleotide hybridization, mutant oligonucleotide hybridization, SSCP, PCR(trademark) and RNase protection. In more specific embodiments, the evaluating is wild-type or mutant oligonucleotide hybridization and the oligonucleotide is configured in an array on a chip or wafer.
Also the present invention provides a method for altering the phenotype of a tumor cell comprising the step of contacting the cell with a tumor suppressor designated PPP2R1B under conditions permitting the uptake of the tumor suppressor by the tumor cell. In particular embodiments, the tumor cell is derived from a tissue selected from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, blood cells, pancreas, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood tissue. More particularly, the phenotype is selected from the group consisting of proliferation, migration, contact inhibition, soft agar growth and cell cycling. In preferred embodiments, the tumor suppressor is encapsulated in a liposome.
Also provided is a method for altering the phenotype of a tumor cell comprising the step of contacting the cell with a nucleic acid (i) encoding a tumor suppressor designated PPP2R1B and (ii) a promoter active in the tumor cell, wherein the promoter is operably linked to the region encoding the tumor suppressor, under conditions permitting the uptake of the nucleic acid by the tumor cell. In specific embodiments, the nucleic acid is a viral vector selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, vaccinia virus and herpesvirus. More particularly, the nucleic acid is encapsulated in a viral particle.
Also contemplated is a method for treating cancer comprising the step of contacting a tumor cell within a subject with a tumor suppressor designated PPP2R1B under conditions permitting the uptake of the tumor suppressor by the tumor cell. In specific embodiments, the subject is a human.
Also provided is a method for treating cancer comprising the step of contacting a tumor cell within a subject with a nucleic acid (i) encoding a tumor suppressor designated PPP2R1B and (ii) a promoter active in the tumor cell, wherein the promoter is operably linked to the region encoding the tumor suppressor, under conditions permitting the uptake of the nucleic acid by the tumor cell.
Another aspect of the present invention contemplates a transgenic mammal in which both copies of the gene encoding PPP2R1B are interrupted or replaced with another gene.
Also provided is a method of determining the stage of cancer comprising the steps of obtaining a sample from a subject; and determining the expression a functional PPP2R1B tumor suppressor in cells of the sample. In certain embodiments, the cancer is a lung cancer. In particular embodiments, the determining comprises assaying for a PPP2R1B nucleic acid or polypeptide in the sample.
Another aspect of the present invention is to provide a method of predicting tumor metastasis comprising the steps of obtaining a sample from a subject; and determining the expression a functional PPP2R1B tumor suppressor in cells of the sample. In specific embodiments, the cancer is distinguished as metastatic and non-metastatic. In particular aspects, the determining comprises assaying for a PPP2R1B nucleic acid or polypeptide in the sample.
Another embodiment provides a method of screening a candidate substance for anti-tumor activity comprising the steps of providing a cell lacking functional PPP2R1B polypeptide; contacting the cell with the candidate substance; and determining the effect of the candidate substance on the cell. In specific embodiments, the cell is a tumor cell. More particularly, the tumor cell has a mutation in the coding region of PPP2R1B. In specific embodiments, the mutation is a deletion mutant, an insertion mutant, a frameshift mutant, a nonsense mutant, a missense mutant or splice mutant. In certain aspects, the determining comprises comparing one or more characteristics of the cell in the presence of the candidate substance with characteristics of a cell in the absence of the candidate substance. More specifically, the characteristic is selected from the group consisting of PPP2R1B expression, phosphatase activity, proliferation, metastasis, contact inhibition, soft agar growth, cell cycle regulation, tumor formation, tumor progression and tissue invasion. In defined embodiments, the candidate substance is a chemotherapeutic or radiotherapeutic agent. In other preferred embodiments, the candidate substance is selected from a small molecule library. In specific embodiments, the cell is contacted in vitro. In alternative embodiments, the cell in contacted in vivo.
Also provided is a method of screening a candidate substance for anti-kinase activity comprising the steps of providing a having PPP2R1B polypeptide comprising at least one serine/threonine kinase site; contacting the cell with the candidate substance; and determining the effect of the candidate substance on the phosphorylation of the site. Similar assays may be performed for anti-phosphatase activity. In specific embodiments, the determining comprises comparing one or more characteristics of the cell in the presence of the candidate substance with characteristics of a cell in the absence of the candidate substance. More particularly, the characteristic is selected from the group consisting of phosphorylation status of PPP2R1B, PPP2R1B expression, phosphatase activity, proliferation, metastasis, contact inhibition, soft agar growth, cell cycle regulation, tumor formation, tumor progression and tissue invasion.
The present invention further provides a method of diagnosing a subject predisposed to cancer comprising the steps of obtaining a sample from a subject; and determining the expression a functional PPP2R1B gene product in cells of the sample. More particularly, the cancer is selected from the group consisting of lung, colon, breast and cervical cancer. In specific embodiments, the cells are selected from the group consisting of breast cells, lung cells, ovarian cells, cervical cells, and endometrial cells. In additional embodiments, the sample is a tissue or fluid sample. Particularly, the determining may comprise assaying for a nucleic acid from the sample. In certain embodiments, the method further comprises subjecting the sample to conditions suitable to amplify the nucleic acid. It is contemplated that the determining may comprise contacting the sample with an antibody that binds immunologically to a PPP2R1B. In certain embodiments, the method further may comprise subjecting proteins of the sample to ELISA. In other embodiments, the method further comprises the step of comparing the expression of PPP2R1B with the expression of PPP2R1B in normal samples. It is contemplated that the comparison may involve evaluating the level of PPP2R1B expression. Alternatively, the comparison may involve evaluating the structure of the PPP2R1B gene, protein or transcript. In specific embodiments, the evaluating is an assay selected from the group consisting of sequencing, wild-type oligonucleotide hybridization, mutant oligonucleotide hybridization, SSCP, PCR(trademark) and RNase protection. More particularly, the evaluating is wild-type or mutant oligonucleotide hybridization and the oligonucleotide is configured in an array on a chip or wafer. In specifically defined embodiments, the sample comprises a mutation in the coding sequence of PPP2R1B.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.